Propagation culture method of fat primitive mesenchymal stem cell

A technology for proliferation and culture of mesenchymal stem cells, applied in the field of proliferation and culture of adipose primitive mesenchymal stem cells, can solve the problems of increasing culture costs, unfavorable purification of stem cells, and unfavorable clinical application of cells, so as to inhibit adherent growth, ensure purity and Effects of dryness and short cell cycle

Inactive Publication Date: 2015-08-12
XIAMEN SINOLEADING PRECISE PHARMA IND DEV CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Chinese patent ZL201210336264.5 discloses a "method for separating and culturing human adipose-derived stem cells and constructing a stem cell bank". When culturing cells, the method takes a long time to change the medium for the first time, which is not conducive to the purification of stem cells. A high concentration of fetal bovine serum is added to the culture medium, which increases the cost of culture, and also introduces exogenous animal protein, which makes the cultured cells unfavorable for clinical application

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  • Propagation culture method of fat primitive mesenchymal stem cell
  • Propagation culture method of fat primitive mesenchymal stem cell
  • Propagation culture method of fat primitive mesenchymal stem cell

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Embodiment 1

[0044] The method for proliferating and culturing adipose-primitive mesenchymal stem cells in this embodiment includes the following steps:

[0045] S1. The isolated single stem cells are divided into 0.5~1×10 4 / cm 2 It was inoculated into a T75 culture flask at a density of 2 Cultured in an incubator with a volume fraction of 5%, the serum-free medium consists of basal medium and supplements, the basal medium is DMEM / F12, and the supplements include: serum substitute 5% (v / v), vitamin C 50ug / ml, human stem cell growth factor 2ng / ml, human platelet-derived growth factor 20ng / ml, L-glutamine 2mmol / ml;

[0046] S2. After 24h of cell culture, change the medium in full (observation of cells under microscope such as figure 1 shown, the cells grow well), remove the residual blood cells and miscellaneous cells, continue to culture, and change the medium in full every three days;

[0047] S3. When the cells reach 80% to 90% confluency ( figure 2 A), pour off the medium in the ...

Embodiment 2

[0054] The method for proliferating and culturing adipose-primitive mesenchymal stem cells in this embodiment includes the following steps:

[0055] S1. The isolated single stem cells are divided into 0.5~1×10 4 / cm 2 It was inoculated into a T75 culture flask at a density of 2 Cultured in an incubator with a volume fraction of 5%, serum-free medium consists of basal medium and supplements, basal medium is DMEM / F12, supplements include: serum substitute 15% (v / v), vitamin C 100ug / ml, human stem cell growth factor 10ng / ml, human platelet-derived growth factor 20ng / ml, L-glutamine 5mmol / ml;

[0056] S2. After culturing the cells for 24 hours, change the medium in full, remove the residual blood cells and miscellaneous cells, continue the culture, and change the medium in full every three days;

[0057] S3. When the cells reach 80% to 90% confluency ( Figure 4 A), pour off the medium in the culture flask, wash the cell surface twice with 0.9% saline, add 10 ml of 0.075%-0.1...

Embodiment 3

[0064] The method for proliferating and culturing adipose-primitive mesenchymal stem cells in this embodiment includes the following steps:

[0065] S1. The isolated single stem cells are divided into 0.5~1×10 4 / cm 2 It was inoculated into a T75 culture flask at a density of 2 Cultured in an incubator with a volume fraction of 5%, serum-free medium consists of basal medium and supplements, basal medium is DMEM / F12, supplements include: serum substitute 2% (v / v), vitamin C 20ug / ml, human stem cell growth factor 0.5ng / ml, human platelet-derived growth factor 5ng / ml, L-glutamine 1mmol / ml;

[0066] S2. After 24 hours of cell culture, change the medium in full to remove residual blood cells and miscellaneous cells, continue to culture, and change the medium in full every three days;

[0067] S3. When the cells reach 80% to 90% confluency ( Image 6 A), pour off the medium in the culture flask, wash the cell surface twice with 0.9% saline, add 10 ml of 0.075%-0.125% (m / v) try...

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Abstract

The invention discloses a propagation culture method of a fat primitive mesenchymal stem cell. The method comprises the following steps: inoculating a separated single stem cell to a culture bottle, adding a serum-free medium, culturing the cells for 24h, and completely replacing the obtained solution for the first time; and continuously culturing the cell by using the serum-free medium. No exogenous proteins are introduced to the serum-free medium, so the cultured cell is helpful for clinic application; and complete solution replacement is carried out after culture for 24h for the first time, so residual parenchyma cells can be fully removed, and purification of the stem cell is facilitated.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to a method for proliferating and culturing adipose primitive mesenchymal stem cells. Background technique [0002] Stem cells are a kind of primitive cells with self-renewal ability and multi-lineage differentiation potential in a suitable microenvironment. Stem cells can be divided into embryonic stem cells, pluripotent stem cells and directed stem cells in various tissues such as hematopoietic stem cells, neural stem cells, etc. At present, there have been experimental reports on the use of stem cells to treat myocardial infarction, lupus erythematosus, rheumatoid arthritis, nerve damage, and muscle atrophy. [0003] Adult stem cells are a primitive stem cell population with multi-lineage differentiation potential that exists in human tissues and can be induced to differentiate into a variety of tissue cells under specific circumstances. Among adult stem cells, mesenchymal stem cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 吴炯
Owner XIAMEN SINOLEADING PRECISE PHARMA IND DEV CO LTD
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